Automated seed sampler and methods of sampling, testing and bulking seeds

ABSTRACT

An automated seed sampler includes a sampling station; a sampler for removing material from a seed in the sampling station; a seed conveyer for conveying the seed from the sampling station to a compartment in a seed tray; and a conveyor for conveying the material removed from the seed to a corresponding compartment in a sample tray. The method of the present invention comprises feeding seeds individually to a sampling station, removing a sample from the seed in the sampling station; conveying the sample to a compartment in a sample tray, and conveying the seed to a corresponding compartment in a seed tray. The samples can be tested, and the seeds can be sorted according to the results of the testing of their corresponding samples.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation application of U.S. patentapplication Ser. No. 11/213,434 filed on Aug. 26, 2005, which claimspriority to U.S. Provisional Application Ser. No. 60/604,604, filed Aug.26, 2004 and U.S. Provisional Application Ser. No. 60/691,100, filedJun. 15, 2005. The entire disclosures of each of these applications areincorporated herein by reference.

BACKGROUND

This invention relates to systems and methods for taking samples frombiological materials such as seeds.

In plant development and improvement, genetic improvements are made inthe plant, either through selective breeding or genetic manipulation,and when a desirable improvement is achieved, a commercial quantity isdeveloped by planting and harvesting seeds over several generations. Notall seeds express the desired traits, and thus these seeds need to beculled from the population. To speed up the process of bulking up thepopulation, statistical samples are taken and tested to cull seeds fromthe population that do not adequately express the desired trait. Howeverthis statistical sampling necessarily allows some seeds without thedesirable trait to remain in the population, and also can inadvertentlyexclude some seeds with the desirable trait from the desired population.

SUMMARY

The present invention relates to systems and methods ofnon-destructively sampling material from seeds. The methods areparticularly adapted for automation, which permits greater sampling thanwas previously practical. With automated, non-destructive samplingpermitted by at least some of the embodiments of this invention, it ispossible to test every seed in the population, and cull those seeds thatdo not express the desired trait. This greatly speeds up the process ofbulking a given seed population, and can result in an improved finalpopulation.

Embodiments of this invention facilitate the testing of most or all ofthe seeds in a population before planting, so that time and resourcesare not wasted in growing plants without the desired traits.

Generally the system of this invention comprises: a sampling station; asampler for removing material from a seed in the sampling station; aseed conveyer for conveying the seed from the sampling station to acompartment in a seed tray; and a conveyor for conveying the materialremoved from the seed to a corresponding compartment in a sample tray.

According to the method of this invention, seeds are fed individually toa sampling station; and held in the sampling station while a sample istaken from the seed. Each sample is conveyed to at least one individualcompartment in a sample tray, and each seed is conveyed to a compartmentin a seed tray with a known relationship with the compartment(s) of thesample tray to which the corresponding sample was conveyed. The samplescan be tested, and the seeds can be sorted based upon the test results.

This system and method of this invention facilitate the automated,non-destructive sampling of seeds. They permit the testing and sortingof large volumes of seeds, thereby facilitating the bulking up of seedpopulations with desirable traits. These and other features andadvantages will be in part apparent, and in part pointed outhereinafter.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a perspective view of a first embodiment of a seed samplersystem constructed according to the principles of this invention;

FIG. 2 is an enlarged perspective view of the seed sampler assembly ofthe seed sampler system;

FIG. 3 is an enlarged perspective view of the hopper and seed feedingmechanism of the seed sampler assembly;

FIG. 4 is a perspective view of the broach for scraping samples from theseeds;

FIG. 5 is a perspective view of the slide for driving the broach;

FIG. 6 is a perspective view of the piston in the feed mechanism of thehopper;

FIG. 7 is a perspective view of a stage with a plurality of seed traysand sample trays mounted thereon;

FIG. 8 is a perspective view of the two-dimensional translationmechanism;

FIG. 9 is a perspective view of the inlet of the seed conveyor;

FIG. 10 is a perspective view of the outlet of the seed conveyor;

FIG. 11 is a perspective view of the outlet of the sample conveyor;

FIG. 12 is a perspective view of the air multiplier used in the seed andsample conveyors;

FIG. 13 is a top plan view of a high throughput seed sampler system inaccordance with the principles of this invention;

FIG. 14 is a side elevation view of the high throughput seed samplersystem;

FIG. 15 is a front perspective view of the seed sampler system;

FIG. 16 is a rear perspective view of the seed sampler system;

FIG. 17 is a perspective view of the sampling station of the highthroughput seed sampler system;

FIG. 18A is a partial perspective view of one portion of the seedsampling station in accordance with the principles of this invention,with the broach retracted;

FIG. 18B is a partial perspective view of one portion of the seedsampling station in accordance with the principles of this invention,with the broach extended;

FIG. 19A is a side elevation view of the seed sampling station, with thebroach in its retracted position;

FIG. 19B is a side elevation view of the seed sampling station, with thebroach in its extended position;

FIG. 20 is a longitudinal cross-sectional view of the seed samplingstation;

FIG. 21 is a front end elevation view of the seed sampling station;

FIG. 22 is a transverse cross-sectional view of the seed samplingstation;

FIG. 23A is a side elevation view of the seed selecting wheel;

FIG. 23B is an exploded view of the seed selecting wheel;

FIG. 23C is a vertical cross sectional view of the seed selecting wheel;

FIG. 24 is a front elevation view of the feeding mechanism;

FIG. 25 is a side elevation view of the feeding mechanism;

FIG. 26A is a perspective view of the feeding mechanism;

FIG. 26B is a side elevation view of the feeding mechanism;

FIG. 26C is a longitudinal cross-sectional view of the feedingmechanism, taken along the plane of line 26C-26C in FIG. 26B;

FIG. 26D is a bottom plan view of the feeding mechanism;

FIG. 27A is an vertical longitudinal cross-sectional view of thesampling mechanism;

FIG. 27B is an enlarged partial vertical cross sectional view of thesampling mechanism as shown in FIG. 27A;

FIG. 28A is a vertical transverse cross-sectional view of the samplingmechanism;

FIG. 28B is a enlarged partial cross-sectional view of the samplingmechanism as shown in FIG. 28A; and

FIG. 29 is an Allelogram depicting maize endosperm tissue samples thathave undergone PCR for detection of a particular SNP polymorphism.

Corresponding reference numerals indicate corresponding parts throughoutthe several views of the drawings.

DETAILED DESCRIPTION

A first embodiment of an automated seed sampler system constructedaccording to the principles of the present invention is indicatedgenerally as 20 in FIG. 1. The seed sampler system 20 is adapted toisolate a seed from a hopper, feed it to a sampling station, scrape asample from the seed, convey the sample to a sample container, andconvey the seed to a corresponding seed container. As shown in FIG. 1,the seed sampler system comprises a support 22, a frame 24 on thesupport; a sampler assembly 26, a stage 28 mounted on a two-dimensionaltranslation mechanism 30, a seed conveyor 32 for transporting seeds fromthe seed sampler assembly, and a sample conveyor 34 for transporting asample removed from a seed to the seed sampler assembly.

As shown in FIG. 1, in the first preferred embodiment the support 22comprises a wheeled cart 40, having a four of vertical posts 42connected by upper and lower longitudinal members 44 and 46, at thefront and back, and upper and lower transverse members 48 and 50 at theleft and right sides, and a table top 52 mounted thereon. A caster 54can be mounted at the bottom of each post 42 to facilitate moving thesupport 22. The details of the construction of the support 22 are notcritical to the invention, and thus the support 22 could have some otherconfiguration without departing from the principles of this invention

As also shown in FIG. 1, the frame 24 comprises four verticallyextending stanchions 60 mounted on the table top 52, which support agenerally horizontal plate 62. The sampler assembly 26 is mounted on theplate 62, as described in more detail below. An arbor 64 is also mountedon the plate, and extends generally horizontally therefrom. The free endof the arbor 64 has first and second vertical posts 66 and 68 formounting a seed conveyor 32 and parts of the sample conveyor 34,respectively. The details of the construction of the frame 24 are notcritical to the invention, and thus the frame could have some otherconfiguration without departing from the principles of this invention.

As shown in FIGS. 1 and 2, the sampler assembly 26 is mounted on theplate 62 of the frame 24. The sample assembly comprises a bin or hopper70, a sampling station 72, and a feed mechanism 74 for delivering asingle seed from the hopper 70 to the sampling station.

As shown in FIGS. 1 and 3, the stage 28 is adapted to securely mount aplurality of seed trays 80 and sample trays 82 in fixed positions andorientations. Each of the seed trays 80 and sample trays 82 ispreferably divided into a plurality of compartments. The number andarrangement of the compartments in the seed trays 80 preferablycorresponds to the number and arrangement of the compartments in thesample trays 82. This facilitates the one-to-one correspondence betweena seed and its sample. However, in some embodiments it may be desirableto provide multiple compartments in the sample tray for each compartmentin the seed tray, for example where multiple tests may be run on thesamples, or where different samples may be taken from the same seed(e.g. samples from different depths).

The stage 28 is mounted on a two-dimensional translation mechanism 30,which in this preferred embodiment comprises a base 90 with a firstlinear actuator 92 having a translatable carriage 94 mounted on a base90, and a second linear actuator 96, having carriage 98 mounted on thecarriage 94 of the first linear actuator 92. The stage 28 is mounted oncarriage 98 of the second linear actuator 96, and thus can be movedprecisely in two dimensions through the operation of the first andsecond linear actuators 92 and 96.

The seed conveyor 32 comprises a tube 100 with an inlet end 102 adjacentthe sampling station 72, and an outlet end 104 mounted on the post 66 ofthe frame 24. There is a first venturi device 106 at the inlet end 102of the tube 100 for inducing an air flow in the tube toward the outletend 104 of the tube, and a second venturi device 108 at the outlet end104 of the tube 100 for inducing an air flow toward the inlet end 102 ofthe tube. The first venturi device 106 is operated to create an air flowin the tube and draw a seed from the sampling station into the tubealong the first end. The second venturi device 108 is then operated tocreate an air flow in the opposite direction, thereby slowing the seeddown to reduce the potential for damaging the seed as it exits theoutlet end 104 of the tube and is delivered to a compartment in thetray. In this preferred embodiment the second venturi 108 actually stopsthe movement of the seed, allowing it to drop under gravity to itscompartment on a tray 80. Various position sensors can be provided onthe tube 100 to detect the presence of the seed, and confirm the properoperation of the seed conveyor 32.

The sample conveyor 34 comprises a tube 120 with an inlet end 122adjacent the sampling station 72, and an outlet end 124 mounted on thepost 68 of the frame 24. There is a first venturi device 126 at theinlet end 122 of the tube 120 for inducing an air flow in the tubetoward the outlet end 124 of the tube. A separator 128 is provided atthe outlet end to separate the sample material from the air streamcarrying it, so that the air stream does not blow the sample out of thecompartment in the tray 82. The separator preferably also contains afilter to prevent cross-contamination of the samples.

As shown in FIG. 2, the seed sampling assembly 26 is adapted to bemounted on the plate 62 on a post 140. The seed sampling assembly 26comprises a hopper mounting plate 142, a slide mounting plate 144 andfour slide standoff supports 146 therebetween. The hopper 70 (shown inFIG. 3), which feeds individual seeds to a sampling station 72, ismounted on the hopper plate 142. The sampling station 72 comprises aseed nest 148 mounted on a nest mount 150, which is supported from theslide mounting plate 144 by a pair of standoffs 152. The nest 148 has arecess opening to its bottom surface, into which the hopper 70 feeds asingle seed. There is a slot in the top of the seed nest 148 throughwhich a portion of a seed in the recess is exposed. A broach 154 (FIG.4) is mounted in a broach holder 156 which is mounted on a slidetransition plate 158 on a programmable slide 160, with a broach clampingblock 162. The programmable slide 160 (FIG. 5) is mounted on theunderside of the slide mounting plate 144, and moves the broach 154through the slot in the seed nest 148 to remove a sample from a seed inthe recess in the seed nest.

As best shown in FIG. 4 the broach 154 has a plurality of teeth 164 thatincrease in height toward the proximal end, so that as the broach 154 isadvanced in the slot, it cuts increasingly deeper into the seed in therecess in the nest 148. The resulting gradual shaving reduces the damageto the seed, protecting its viability. Moreover, as described in moredetail below, by cutting at different depths at different times, samplesfrom different depths of the same seed can be separated for separateanalysis.

A sample transfer tube 166 extends from the recess in the seed nest 148,and has a connector 168 on its end for connection to the sample conveyor34.

The sampling station 26 also includes a hopper 70, shown best in FIG. 3.The hopper 70 comprises left and right hopper mounting plates 170 and172, and a cylinder mounting plate 174 and a upper cylinder bracket 176.The hopper 70 also has a front panel 178, a back panel 180, first andsecond end panels 182 and 184, and bottom 186. A divider 188 divides thehopper into first and second compartments 190 and 192. The firstcompartment 190 holds a supply of seeds which are individuallytransferred to the second compartment 192.

A piston actuator 194 operates a piston 196 to lift a seed out of thefirst compartment. A air jet assembly 198 transfers a seed from the endof the piston 196 to the second compartment 192. The second compartmenthas a shaped bottom 200, with a well 202 for receiving the seed andpositioning it. A piston actuator 210 operates a piston 214 to lift aseed out of the second compartment 192. An air jet assembly 216 is usedto stir the seeds during the seed pick up procedure.

As shown in FIG. 7, the stage 28 has brackets 220 for mounting seedtrays 80 and sample trays 82 in registration so that the seed conveyorand the sample conveyor deliver seeds and samples to correspondingcompartments, in the respective trays. The sample trays 82 can (asshown) be adapted to hold individual vials. Of course, trays ofdifferent configurations could be used, for example where multiplecompartments are provided for multiple samples from the same seed. Forexample where one sample is divided into several samples, or where thesamples are separated from where they are taken, e.g. by depth.

As shown in FIG. 8, the two-dimensional translation mechanism 30 alsoincludes a slider 230 having a rail 232 and a carriage 234, that ispositioned parallel to the first linear actuator 92. The second linearactuator 96 is mounted on the carriage 94 having carriage 98 mounted onthe carriage 94 of the first linear actuator 92. The stage 28 is mountedon carriage 98 of the second linear actuator 96, and thus can be movedprecisely in two dimensions through the operation of the first andsecond linear actuators 92 and 96. Under appropriate control thetranslation mechanism can align individual compartments of the seedtrays 80 and sample trays 82 with the outlets of the seed conveyor andsample conveyer.

As shown in FIG. 9, at the inlet end 102 of the tube 100 of seedconveyor 32, a bracket 240 mounts an air amplifier 242 and a seed sensortube 244. The bracket 240 comprises sections 246, 248, 250, 252 and 254.As shown in FIG. 2, the bracket 240 is mounted on the hopper mountingplate 142. The air amplifier 242 (shown in FIG. 12) is adapted to beconnected to a source of compressed air. When air is applied to the airamplifier, it induces an air flow through the tube 100, employing theventuri effect. The sensor tube 244 carries seed sensors 256 for sensingthe passage of a seed therethrough. The sensors 256 are preferablyoptical sensors aligned with openings in the sensor tube 244 whichoptically detect the passage of a seed.

As shown in FIG. 10, a seed discharge assembly 260 is disposed at theoutlet end 104 of the tube 100 of seed conveyor 32. The dischargeassembly is mounted on post 66, with a bracket 262 and a dischargesupport 264. A seed sensor tube 266 is mounted in the bracket 262, andcarries seed sensors 268 for sensing the passage of a seed therethrough.The sensors 268 are preferably optical sensors aligned with openings inthe sensor tube 266 which optically detect the passage of a seed. An airamplifier 270 is connected to the seed sensor tube 266. The airamplifier 270 (FIG. 12) is adapted to be connected to a source ofcompressed air. When air is applied to the air amplifier, it induces anair flow through the tube 100, employing the venturi effect. Below theair amplifier 270 is a connector tube 272, and below that is a ventedseed discharge tube 274, which is also supported by a seed dischargetube holder 276, carried on a seed discharge tube actuator 278.

The inlet end 122 of the tube 120 of the sample conveyor 34 is connectedvia connector 168 to the sample discharge tube 166. As shown in FIG. 11,the outlet end 124 of the tube 120 is connected to a sample connector280, which in turn is connected to air amplifier 282, which is connectedto chip nozzle assembly 284. The chip nozzle assembly 284 is mounted onthe seed discharge tube holder 286, which is carried on a dischargeactuator 288. The discharge actuator is mounted on the post 68. Filters290 are mounted on the outlets of the chip nozzle assembly 284, toprevent samples being discharged from contaminating the othercompartments.

Operation of the Sampler System

In operation, a plurality of seeds, for example soybeans, are depositedin the hopper 70. The seed feed mechanism 74 conveys an individual seedto the sampling station 72. At the sampling station, a sample ofmaterial is removed from the seed in a manner that minimizes the impactto the viability of the seed.

The sample is removed from the sampling station 72 by the sampleconveyor 34. The venturi device 126 creates an air flow in the tube 120toward the outlet end 124. The sample material is drawn into the tubeand toward the compartment of the sample tray aligned with outlet end124 of the tube 120. The separator 128 separates the sample from the airstream carrying it, and allows the sample to drop into the compartment.In some embodiments, the sample may be distributed to two or morecompartments in the sample tray, in which case the two-dimensionaltranslation mechanism 30 is operated to bring one or more additionalcompartments into alignment with the outlet 124. It is possible toaccurately coordinate the movement of the sample trays with theoperation of the sampling station 72 so that samples from differentportions of the seed, and in particular different depths of the seed,can be delivered to separate compartments in the sample tray.

After the sampling from the seed is completed, the seed conveyor 32 isoperated to remove the seed from the sampling station. The first venturidevice 106 is operated to create an air flow in the tube and draw a seedfrom the sampling station 72 into the tube 100. The second venturidevice 108 is then operated to create an air flow in the oppositedirection, thereby slowing the seed down to reduce damage to the seed asit exits the outlet end 104 of the tube 100 and is delivered to acompartment in the seed tray 82. The second venturi 108 preferably stopsthe movement of the seed, allowing it to drop under gravity to itscompartment on a tray 80. The operation of the first and second venturis106 and 108 can be timed, or they can be triggered by position sensorsmonitoring the tube 100.

An embodiment of a high throughput seed sampler system is indicatedgenerally as 500 in FIGS. 13-26. As shown in FIGS. 13 and 14, the seedsampler system 500 comprises a sampling station 502, a sample handlingstation 504, and a seed handling station 506. It is desirable, but notessential, that the seed sampler system 500 fit on one or more wheeledcarts that can pass though conventional doorways, so that the system canbe conveniently transported. In this preferred embodiment, the seedsampling station 502 is mounted on a cart 508, the sample handlingstation is mounted on a cart 510, and the seed handling station ismounted on a cart 512.

The seed sampling station 502 comprises a seed feeder 514 and a seedchipper 516. A plurality of columns 518 extend vertically upwardly fromthe surface 520 of the cart 508. A platform 522 is mounted on top ofcolumns 518 and supports the seed chipper 514. Two L-brackets 524 extendhorizontally from the columns 518, and support a platform 526. A stage528 is mounted on the platform 526 by a plurality of posts 530 andsupports the seed feeder 514.

A plurality of pillars 532 extend upwardly from the plate 522. A plate534 is mounted on the pillars 532. A plurality of posts 536 depend fromthe plate 534, and support a shelf 538.

As shown in FIGS. 13, 14, 15 and 16, the seed feeder 514 comprises ahopper 550, with a shaped surface adapted to feed seeds deposited intothe hopper toward a separating wheel 552 (see also FIGS. 23A through23C). The separating wheel 552 is mounted for rotation in a verticalplane adjacent the hopper 550, and has a plurality of spaced recesses554 each having an opening 556 therein communicating with a vacuumsystem (not shown). The wheel 552 is advanced with an indexing motor560. Individual seeds are picked up by the recesses 554 in the wheel 552and held in the recesses by suction from the vacuum system via openings556. A wiper 562 wipes individual seeds from the recesses 554, allowingthem to drop through a guide 564 into an opening in a distributor 566.

As shown in FIGS. 24-26, the distributor 566 comprises a shaft 568having a plurality (six in the preferred embodiment) of passages 570extending transversely there through. Sleeves 572 and 574 are slidablymounted over each end of the shaft 568 to translate between first(inboard) and second (outboard) positions. The sleeves 572 and 574 havea plurality of pairs of aligned openings 576 and 578 on opposite sidesthereof. The openings 576 are elongate, and the openings 576 and 578 aresized and arranged so that when the sleeves 572 and 574 are in theirfirst (inboard) position (on the left side in FIG. 24), a portion of theelongate openings 576 is aligned with a passage 570 in the shaft 568,and when the sleeves are in their second (outboard) positions a portionof the elongate openings 576 and the second openings 578 are alignedwith the passage (on the right side in FIG. 24). An actuator 580selectively slides the sleeves 572 and 574 between their first andsecond positions.

The distributor 566 is mounted by a bracket 582 on the carriage 584 of alinear actuator 586, to translate relative to the guide 564,successively bringing each of the passages 570 in the shaft 568 intoalignment with the guide 564 so that a seed can be deposited therein. Aseed sensor (not shown) can be mounted adjacent the guide 564 to confirmthat a seed is deposited in each passage 570. A plurality of air nozzles590 are mounted on the stage 528, and are aligned with the passages 570when the distributor 566 is moved to its dispensing position by theactuator 586. A tube 592 is aligned with each passage 570, and each tubeconnects to one of a plurality of seed sampling stations 600 in the seedchipper 516. The sleeves 572 and 574 are translated allowing the seedsin the passages 570 to drop into tubes 592. One of the nozzles 590 isaligned with each of the passages 570, and is actuated to facilitate themovement of the seeds from the passages 570 through the tubes 592 totheir respective seed sampling stations 600.

There is preferably a port 596 through the hopper 550 that aligns withthe opening 556 in each recess 554 as the wheel 552 turns. The port 596can be connected to a vacuum to draw any dirt or pieces of seed husks orseed that might clog the openings 556 in the recesses 554, and impairthe ability of the wheel 552 to select individual seeds from the hopper550.

The seed chipper 516 comprises at least one, and in this preferredembodiment six, sampling stations 600. Each seed sampling station 600removes a sample of material from a seed delivered to it. In thispreferred embodiment the sampling stations 600 are arranged or ganged intwo groups of three, but the number and arrangement of the samplingstations could vary. The sample handling station 504 receives tissuesamples removed from a seed and transported away from each samplingstation 600. Similarly, the seed handling station 506 receives a seedafter the sample has been removed from the seed, and the seed istransported from the sampling station 600.

Each seed sampling station 600 has an inlet collar 602 connected to thetube 592, that opens to a chamber 604. The bottom surface of the chamber604 is formed by the end of a rod 606 of actuator 608. The surface ofthe bottom is below the inlet collar 602 to ensure that the entire seeddrops into the chamber 604 and is not caught in a position only partlyin the chamber. A vent 610 may be positioned opposite from the inletcollar 602 to allow air from air nozzles 590 to escape. The vent 610 canbe covered with a mesh grille 612 to prevent the seed from escaping thechamber 604 and to cushion the seed as it is delivered into the chamber.

This rod 606 lifts a seed out of the chamber 604 and into aseed-receiving recess 614 in the underside of a seed sampling plate 616.The sampling plate 616 has a sampling opening 618 through which a seedin the seed-receiving recess 614 protrudes. A sampling groove 620 isformed in the top surface of the sampling plate 616 such that a portionof a seed in the recess 614 protrudes into the groove. The samplingplate 616 also has laterally oriented openings 622 and 624 thereinaligned with the seed-receiving recess 614. When the rod 606 lifts aseed delivered to the sampling station 600 into the recess 614 in theplate 616, fingers 626 and 628 extend transversely through the openings622 and 624 and are operated by actuator 630 to engage and compress theseed. It has been discovered that compressing at least certain types ofseeds during the sampling process can improve viability of the seedsafter sampling. For seeds such as soybean seeds, it has been found thata compressive pressure enhances seed viability, and that compressivepressure of between about 2.5 and about 5 pounds is sufficient toenhance viability.

A sampling broach 650 having a plurality of cutting edges 652reciprocates in the groove 620 so that the cutting edges 652 can scrapea sample from a seed being held in the recess 614 by the rod 606 and thefingers 626 and 628. The cutting edges 652 are preferably parallel, andoriented an oblique angle less than 90° relative the direction of travelof the broach. It is desirable, but not essential, that the cuttingedges 652 be angled sufficiently that one edge remains in contact withthe seed at all time. Angling the cutting edges allows the next blade toestablish contact with the seed before the current blade loses contactwith the seed. In the preferred embodiment the cutting edges areoriented at an angle of about 60°, although this angle will dependsomewhat upon the width of the broach. The width of the broach can alsobe important to preserving seed viability after sampling, and may varydepending upon the type of seed and its moisture content.

The cutting edges 652 are staggered, each cutting progressively deeperthan the previous. The amount of sample material and the depth of thecut can be controlled by controlling the advancement of the broach 650.For smaller samples and shallower depths of cut, the stroke of thebroach 650 is shorter, and for larger samples or deeper depths of cut,the stroke of the broach is longer. For partial strokes, tissue from theseed may be trapped between edges 652. The broach 650 can be advancedand retracted to help release all of the sample. For example, after theseed is released, the broach may be advanced and retracted to helpremove seed tissue trapped between the cutting edges. The full range oftravel of the broach 650 is shown in FIGS. 19A and 19B.

The sampling broach 650 is preferably driven by a linear actuator 654.In the preferred embodiment, three broaches 650 are driven by a singleactuator 654. Using a single actuator to operate multiple broaches savesspace and is more economical.

A sample transport system 656 comprising a conduit 658 having an inlet660 communicating with a passage 662 that opens to the sampling opening618 and the groove 620 in the sampling plate 616 removes tissue samplesmade by the action of the cutting edges 652 of the sampling broach 650.The conduit 658 transports the sample to outlet 664 where it isdeposited in a unique sample holder in the sample handling station 504.This sample holder may be, for example, a well 666 in a tray 668 mountedon a x-y indexing table 670 on cart 510, so that the relationshipbetween samples and their respective seeds can be determined. The sampletransport system 656 includes an air jet 672 which induces air flowthrough the conduit 658 to move the sample through the conduit.

A second sampling mechanism can be mounted on the linear actuator 654and moves with the broach 650. The second sampling mechanism cancomprise a coring device 674 having a coring tool 676 for taking a plugsample of the seed from the kerf made by the broach 650. This tissue inthis sample is from a deeper location than the tissue scraped by thebroach 650, and provides different information. In some embodiments thematerial removed by the broach 650 might simply be discarded, and onlythe sample taken with the coring device 674 retained. In someembodiments both samples may be retained and separately stored forseparate testing. In still other embodiments the only sample is thesample removed by the broach 650. In embodiments without the secondsampling mechanism, the coring device 674 and coring tool 676 can bereplaced with an actuator with a simple push rod that extends throughthe sampling opening 618 to help push a seed in the recess 614.

A seed transport system 680 having an inlet 682 adjacent recess 614 fordrawing in seeds after they are released by the fingers 626 and 628 andthe rod 606 lowers the seed after the sampling operation. The seedtransport system 680 transports the seeds to a unique seed holder in theseed handling station 506 on the cart 512. This seed holder may be, forexample, a well 684 in a tray 686 mounted on an x-y indexing table 688on cart 612, so that the relationship between samples and theirrespective seeds can be determined. The seed transport mechanism 680includes an air jet 690 which induces air flow through the conduit 680to move the sample through the conduit.

Operation

In operation, a plurality of seeds, e.g. soybean seeds, are dumped intothe hopper 550 of the sampling system 500. These seeds flow undergravity toward the disk 552, suction through the ports 556 hold one seedin each cavity 554. As the disk 552 is rotated by the indexing motor560, individual seeds are wiped from the disk by the wiper 562, and fallunder gravity through the guide 564 to the outlet. The linear actuator586 moves the distributor 566 so that each passage 570 of thedistributor aligns with the guide 564 to load one seed through theopening 576 and into passage 570. When all of the passages 570 in thedistributor 566 are full, the linear actuator 586 moves the distributorinto position to load its seeds into sampling stations 600 in the seedchipper 516. The sleeves 572 and 574 are moved by actuator 580, whichaligns the openings 578 with the passages 570, allowing the seeds in thepassages 570 to fall into the tubes 592 that lead to the sampling units600. The nozzles 590 provide a blast of air that helps urge the seedsfrom the passages 570 through the tubes 592 to the chambers 604 in thesampling units 600.

Preferably all of the passages 570 are loaded in series and dischargetheir seeds simultaneously to the sampling units 600, but thedistributor could be programmed to operate in some other manner. Oncethe seeds arrive in the sampling stations 600, the rods 606 lift theseeds into the recesses 614 in the underside of the plates 616. Therecesses 614 may be sized and shaped to help optimally orient the seed.In the recesses 614, a portion of the seeds protrude through thesampling holes 618 and into the grooves 620. The broaches 650 aretranslated in the grooves 620, allowing their cutting edges 652 toremove material from the portions of the seeds protruding into thegrooves 620, and forming small kerfs in the seeds. As each broach 650removes material, the sample transport system 656 draws the samplematerial through passage 662 and into the inlet 660. The samples travelin conduits 658 away from the sampling stations 600 to a sample storagelocation, such as wells 666 in a sample tray 668. A second sample can betaken by the coring tool 676 of sampling device 674 through the opening618 in the sampling plate 616. After the sampling is completed, the rod606 retracts, and as the seed drops the sampled-seed transport system680 transports the sampled seed to a seed storage location, such as awell 684 in a seed tray 686.

The indexing tables 670 and 688 move to align different wells with theoutlets of the sample transport system 656 and the seed transport system680, and the sample process is repeated. When all of the wells 666 in asample tray 668 are full, the samples in the sample tray can be tested,and the seeds in the corresponding seed tray 686 can be selected basedupon the results of the testing of samples. The sampling preferably doesnot substantially adversely affect the viability of the seeds.

Applications

The present invention provides methods for analyzing seeds having adesired trait, marker or genotype. In one aspect of the invention, theanalytical methods allow individual seeds to be analyzed that arepresent in a batch or a bulk population of seeds such that the chemicaland/or genetic characteristics of the individual seeds can bedetermined.

Samples prepared by the present invention can be used for determining awide variety of physical, chemical and/or genetic traits. Examples ofchemical analyses for use in the methods of the present inventioninclude starch content, protein content, oil content, determination offatty acid profiles, etc.

In one embodiment, the methods and devices of the present invention canbe used in a breeding program to select plants or seeds having a desiredtrait or marker genotype. The methods of the present invention can beused in combination with any breeding methodology and can be used toselect a single generation or to select multiple generations. The choiceof breeding method depends on the mode of plant reproduction, theheritability of the trait(s) being improved, and the type of cultivarused commercially (e.g., F₁ hybrid cultivar, pureline cultivar, etc).Selected, non-limiting approaches for breeding the plants of the presentinvention are set forth below. It is further understood that anycommercial and non-commercial cultivars can be utilized in a breedingprogram. Factors such as, for example, emergence vigor, vegetativevigor, stress tolerance, disease resistance, branching, flowering, seedset, seed size, seed density, standability, and threshability etc. willgenerally dictate the choice.

In a particular embodiment, the methods of the present invention areused to determine the genetic characteristics of seeds in amarker-assisted breeding program. Such methods allow for improvedmarker-assisted breeding programs wherein nondestructive direct seedsampling can be conducted while maintaining the identity of individualsfrom the seed sampler to the field. As a result, the marker-assistedbreeding program results in a “high-throughput” platform wherein apopulation of seeds having a desired trait, marker or genotype can bemore effectively bulked in a shorter period of time, with less field andlabor resources required. Such advantages will be more fully describedbelow.

In one embodiment, the present invention provides a method for analyzingindividual seeds within a population of seeds having geneticdifferences. The method comprises removing a sample comprising cellswith DNA from seeds in the population without affecting the germinationviability of the seeds; screening the DNA extracted from the sample forthe presence or absence of at least one genetic marker; selecting seedsfrom the population based upon the results of the DNA screening; andcultivating plants from the selected seed.

As described above, the sampling systems and methods of this inventionprotect germination viability of the seeds so as to be non-destructive.Germination viability means that a predominant number of sampled seeds,(i.e, greater than 50% of all sampled seeds) remain viable aftersampling. In a particular embodiment, at least about 75% of sampledseeds, and in some embodiments at least about 85% of sampled seedsremain viable. It should be noted that lower rates of germinationviability may be tolerable under certain circumstances or for certainapplications, for example, as genotyping costs decrease with timebecause a greater number of seeds could be sampled for the same genotypecost.

In another embodiment, germination viability is maintained for at leastabout six months after sampling to ensure that the sampled seed will beviable until it reaches the field for planting. In a particularembodiment, the methods of the present invention further comprisetreating the sampled seeds to maintain germination viability. Suchtreatment may generally include any means known in the art forprotecting a seed from environmental conditions while in storage ortransport. For example, in one embodiment, the sampled seeds may betreated with a polymer and/or a fungicide to protect the sampled seedwhile in storage or in transport to the field before planting.

DNA may be extracted from the sample using any DNA extraction methodsknown to those of skill in the art which will provide sufficient DNAyield, DNA quality, and PCR response. A non-limiting example of suitableDNA-extraction methods is SDS-based extraction with centrifugation. Inaddition, the extracted DNA may be amplified after extraction using anyamplification method known to those skilled in the art. For example, onesuitable amplification method is the GenomiPhi® DNA amplification prepfrom Amersham Biosciences.

The extracted DNA is screened for the presence or absence of a suitablegenetic marker. A wide variety of genetic markers are available andknown to those of skill in the art. The DNA screening for the presenceor absence of the genetic marker can be used for the selection of seedsin a breeding population. The screening may be used to select forquantitative trait loci (QTL), alleles, or genomic regions (haplotypes).The alleles, QTL, or haplotypes to be selected for can be identifiedusing newer techniques of molecular biology with modifications ofclassical breeding strategies.

In one embodiment, the seed is selected based on the presence or absenceof a genetic marker that is genetically linked with a QTL. Examples ofQTLs which are often of interest include but are not limited to yield,lodging resistance, height, maturity, disease resistance, pestresistance, resistance to nutrient deficiency, and grain composition.Alternatively, the seed can be selected based on the presence or absenceof a marker that is genetically linked with a haplotype associated witha QTL. Examples of such QTL may again include without limitation yield,lodging resistance, height, maturity, disease resistance, pestresistance, resistance to nutrient deficiency, and grain composition.

Selection of a breeding population could be initiated as early as the F₂breeding level, if homozygous inbred parents are used in the initialbreeding cross. An F₁ generation could also be sampled and advanced ifone or more of the parents of the cross are heterozygous for the allelesor markers of interest. The breeder may screen an F₂ population toretrieve the marker genotype of every individual in the population.Initial population sizes, limited only by the number of available seedsfor screening, can be adjusted to meet the desired probability ofsuccessfully identifying the desired number of individuals. See Sedcole,J. R. “Number of plants necessary to recover a trait.” Crop Sci.17:667-68 (1977). Accordingly, the probability of finding the desiredgenotype, the initial population size, and the targeted resultingpopulation size can be modified for various breeding methodologies andinbreeding level of the sampled population.

The selected seeds may be bulked or kept separate depending on thebreeding methodology and target. For example, when a breeder isscreening an F₂ population for disease resistance, all individuals withthe desired genotype may be bulked and planted in the breeding nursery.Conversely, if multiple QTL with varying effects for a trait such asgrain yield are being selected from a given population, the breeder maykeep individual identity preserved, going to the field to differentiateindividuals with various combinations of the target QTL.

Several methods of preserving single seed identity can be used whiletransferring seed from the chipping lab to the field. Methods include,but are not limited to, transferring selected individuals to seed tape,a cassette tray, or indexing tray, transplanting with peat pots, andhand-planting from individual seed packets.

Multiple cycles of selection can be utilized depending on breedingtargets and genetic complexity.

Advantages of using the screening methods of this invention include,without limitation, reduction of labor and field resources required perpopulation or breeding line, increased capacity to evaluate a largernumber of breeding populations per field unit, and increased capacity toscreen breeding populations for desired traits prior to planting. Fieldresources per population are reduced by limiting the field spacerequired to advance the desired genotypes. For example, a population of1,000 individuals may be planted at 25 seeds per row consuming a totalof 40 rows in the field. Using conventional tissue sampling, all 1,000plants would be tagged and manually sampled by scoring leaf tissue.Molecular marker results would be needed prior to pollination and onlythose plants containing the desired genetic composition would bepollinated. Thus, if it was determined that 50 seeds contained thedesired genetic composition, conventional breeding methodology wouldhave required the planting of 1000 plants to obtain 50 seeds. Bycontrast, the screening methods of this invention allow the breeder toscreen the 1,000 seeds in the lab and select the 50 desired seeds priorto planting. The 50 individuals can then be planted in the field,consuming only two 25 seed rows. Additionally, the screening methods ofthis invention do not require tagging or sampling in the field, therebysignificantly reducing the required manual labor resources.

In addition to reducing the number of field rows per population, thescreening methods of this invention may further increase the number ofpopulations the breeder can evaluate in a given breeding nursery. Usingthe above example wherein 50 seeds out of each population of 1000 seedscontained the desired genetic composition, a breeder applying themethods of this invention could evaluate 20 populations of 50 seeds eachusing the same field area consumed by a single population usingconventional field tissue sampling techniques. Even if the populationsare selected for a single allele, using a 1:2:1 expected segregationratio for an F₂ population, the breeder could evaluate 4 populations inthe same field area as a single field tissue sampled population.

A potential further advantage to seed chipping is that it could be usedto mitigate the risks associated with growing plants in certaingeographies where plants may grow poorly or experience poorenvironmental conditions, or may even be destroyed during storms. Forexample, seeds with the “best” genotype or marker composition could beplanted in geography 1 and seeds with the “next best” genotype could beplanted in geography 2. In this case geography 2 would be a backup incase any problem befell the plants grown in geography 1. This is verydifficult to do with the traditional method of taking tissue samplesfrom germinated plants for genotyping, because these plants would thenneed to be uprooted and transplanted to the second geography. Using themethods of this invention avoids the problem of transplantation.

The screening methods of the invention may further be used in a breedingprogram for introgressing a trait into a plant. Such methods compriseremoving a sample comprising cells with DNA from seeds in a population,screening the DNA extracted from each seed for the presence or absenceof at least one genetic marker, selecting seeds from the populationbased upon the results of the DNA screening; cultivating a fertile plantfrom the seed; and utilizing the fertile plant as either a female parentor male parent in a cross with another plant.

Examples of genetic screening to select seeds for trait integrationinclude, without limitation, identification of high recurrent parentallele frequencies, tracking of transgenes of interest or screening forthe absence of unwanted transgenes, selection of hybrid testing seed,and zygosity testing.

The identification of high recurrent pair allele frequencies via thescreening methods of the present invention again allows for a reducednumber of rows per population and an increased number of populations, orinbred lines, to be planted in a given field unit. Thus, the screeningmethods of the present invention may also effectively reduce theresources required to complete the conversion of inbred lines.

The methods of the present invention further provide quality assurance(QA) and quality control by assuring that regulated or unwantedtransgenes are identified and discarded prior to planting. Thisapplication in a QA capacity could effectively eliminate unintentionalrelease infractions.

The methods of the present invention may be further applied to identifyhybrid seed for transgene testing. For example, in a conversion of aninbred line at the BCnF₁ stage, a breeder could effectively create ahybrid seed lot (barring gamete selection) that was 50% hemizygous forthe trait of interest and 50% homozygous for the lack of the trait inorder to generate hybrid seed for testing. The breeder could then screenall F₁ seeds produced in the test cross and identify and select thoseseeds that were hemizygous. Such method is advantageous in thatinferences from the hybrid trials would represent commercial hybridgenetics with regard to trait zygosity.

Other applications of the screening methods of this invention foridentifying and tracking traits of interest carry the same advantagesidentified above with respect to required field and labor resources.Generally, transgenic conversion programs are executed in multi-seasonlocations which carry a much higher land and management cost structure.As such, the impact of either reducing the row needs per population orincreasing the number of populations within a given field unit aresignificantly more dramatic on a cost basis versus temperateapplications.

Still further, the screening methods of this invention may be used toimprove the efficiency of the doubled haploid program through selectionof desired genotypes at the haploid stage and identification of ploidylevel to eliminate non-haploid seeds from being processed and advancingto the field. Both applications again result in the reduction of fieldresources per population and the capability to evaluate a larger numberof populations within a given field unit.

In another embodiment, the invention further provides an assay forpredicting embryo zygosity for a particular gene of interest (GOI). Theassay predicts embryo zygosity based on the ratio of the relative copynumbers of a GOI and of an internal control (IC) gene per cell or pergenome. Generally, this assay uses an IC gene that is of known zygosity,e.g., homozygous at the locus (two IC copies per diploid cell), fornormalizing measurement of the GOI. The ratio of the relative copynumbers of the IC to the GOI predicts the GOI copy number in the cell.In a homozygous cell, for any given gene (or unique genetic sequence),the gene copy number is equal to the cell's ploidy level since thesequence is present at the same locus in all homologous chromosomes.When a cell is heterozygous for a particular gene, the gene copy numberwill be lower than the cell's ploidy level. The zygosity of a cell atany locus can thus be determined by the gene copy number in the cell.

In a particular embodiment, the invention provides an assay forpredicting corn embryo zygosity. In corn seed, the endosperm tissue istriploid, whereas the embryo tissue is diploid. Endosperm that ishomozygous for the IC will contain three IC copies. Endosperm GOI copynumber can range from 0 (homozygous negative) to 3 (homozygouspositive); and endosperm GOI copy number of 1 or 2 is found in seedheterozygous for the GOI (or hemizygous for the GOI if the GOI is atransgene). Endosperm copy number is reflective of the zygosity of theembryo: a homozygous (positive or negative) endosperm accompanies ahomozygous embryo, heterozygous endosperm (whether a GOI copy number of1 or 2) reflects a heterozygous (GOI copy number of 1) embryo. Theendosperm GOI copy number (which can range from 0 to 3 copies) can bedetermined from the ratio of endosperm IC copy number to endosperm GOIcopy number (which can range from 0/3 to 3/3, that is, from 0 to 1),which can then be used to predict zygosity of the embryo.

Copy numbers of the GOI or of the IC can be determined by any convenientassay technique for quantification of copy numbers, as is known in theart. Examples of suitable assays include, but are not limited to, RealTime (TaqMan®) PCR (Applied Biosystems, Foster City, Calif.) andInvader® (Third Wave Technologies, Madison, Wis.) assays. Preferably,such assays are developed in such a way that the amplificationefficiency of both the IC and GOI sequences are equal or very similar.For example, in a Real Time TaqMan® PCR assay, the signal from asingle-copy GOI (the source cell is determined to be heterozygous forthe GOI) will be detected one amplification cycle later than the signalfrom a two-copy IC, because the amount of the GOI is half that of theIC. For the same heterozygous sample, an Invader® assay would measure aGOI/IC ratio of about 1:2 or 0.5. For a sample that is homozygous forboth the GOI and the IC, the GOI signal would be detected at the sametime as the IC signal (TaqMan®), and the Invader assay would measure aGOI/IC ratio of about 2:2 or 1.

These guidelines apply to any polyploid cell, or to haploid cells (suchas pollen cells), since the copy number of the GOI or of the IC remainproportional to the genome copy number (or ploidy level) of the cell.Thus, these zygosity assays can be performed on triploid tissues such ascorn endosperm.

EXAMPLES

The following examples are merely illustrative, and not limiting to thisdisclosure in any way.

Example 1

This example describes an assay for predicting the zygosity of cornembryos using an internal control (IC) gene homozygous at the locus(i.e., two IC copies in the diploid embryo and three IC copies in thetriploid endosperm). In an inbred line of a diploid (or higher ploidy)organism such as corn, the endogenous internal control is typicallyhomozygous; transgenic events in such organisms at the first generation(termed “R0” in corn) are typically hemizygous (that is, the transgeneis typically present in only one of the two or more homologouschromosomes). Corn (Zea mays) is a diploid organism, thus a “singlecopy” R0 event has one copy of the GOI per cell, but 0.5 copies perhaploid genome, a “two copy” R0 event has two copies of the GOI percell, but 1 copy per haploid genome, and so forth.

In this example, tubulin was used as the IC gene, and the GOI was atransgene encoding neomycin phosphotransferase II (NPT II), which isused for kanamycin resistance selection. Endosperm (triploid) tissue wastaken from seed (either by hand sampling or by scraping a seed with anautomated sampler of the present invention). The endosperm-sampled seedwas germinated, and leaf tissue (diploid) from successfully germinatedplants was also sampled for genetic analysis. The leaf tissue correlatesdirectly with embryo zygosity and was thus used to demonstrate thatendosperm zygosity generally predicted zygosity of the embryo and toconfirm homozygosity calls from the endosperm. Total genomic DNA wasextracted from endosperm tissue and from leaf tissue, and quantitativelyanalyzed using an Invader® assay with oligonucleotide probes specificfor the gene of interest, NPT II, or for the internal control gene,tubulin. The ratio of the GOI to IC was measured using conventionalmolecular biology techniques. See Table 1. A summary of results ofmultiple experiments are shown in Table 2.

Results indicated that endosperm zygosity generally predicted zygosityof the embryo (as indicated by the leaf zygosity) and was reliable inpredicting homozygosity for all seeds that germinated. Furthermore,endosperm zygosity analysis gave few false-negative homozygouspredictions (especially when the endosperm tissue was obtained with theautomated sampler). These results demonstrate that for a cell of a knownploidy level, the ratio of copy number of a GOI to that of an ICindicates the zygosity of that cell. Furthermore, the zygosity assay ofthe present invention can predict zygosity of one tissue based on thezygosity of another, that is, the assay can predict the embryo zygositybased on the endosperm zygosity.

TABLE 1 Automated Manual Ratio Automated Zygosity Ratio Manual Zygosity1.39 Heterozygous 1.42 Heterozygous 0.14 neg homozygous 0.12 neghomozygous 0.08 neg homozygous 0.08 neg homozygous 0.13 neg homozygous0.10 neg homozygous 0.10 neg homozygous 0.08 neg homozygous 1.55Heterozygous 1.38 Heterozygous 0.84 Heterozygous 1.45 Heterozygous 0.14neg homozygous 1.48 Heterozygous 1.48 Heterozygous 1.37 Heterozygous1.39 Heterozygous 1.47 Heterozygous 2.03 POS homozygous 1.93 POShomozygous 0.13 neg homozygous 0.05 neg homozygous 1.71 inconclusive1.81 POS homozygous 0.81 Heterozygous 1.41 Heterozygous 1.84 POShomozygous 1.77 POS homozygous 1.54 Heterozygous 1.43 Heterozygous 1.48Heterozygous 1.50 Heterozygous 0.92 Heterozygous 1.40 Heterozygous 1.51Heterozygous 1.42 Heterozygous 1.60 Heterozygous 1.37 Heterozygous 0.86Heterozygous 1.47 Heterozygous 1.81 POS homozygous 2.02 POS homozygous0.15 neg homozygous Low DNA 1.89 POS homozygous 1.85 POS homozygous 0.21neg homozygous 0.10 neg homozygous 0.09 neg homozygous 0.11 neghomozygous 0.89 Heterozygous 1.50 Heterozygous 1.50 Heterozygous 1.37Heterozygous 1.82 inconclusive 2.02 POS homozygous 2.14 POS homozygous0.99 inconclusive 1.22 Heterozygous 1.44 Heterozygous 2.22 POShomozygous 2.24 POS homozygous 0.79 Heterozygous 1.40 Heterozygous 1.23Heterozygous 1.47 Heterozygous 1.49 Heterozygous 1.38 Heterozygous 1.33Heterozygous 1.37 Heterozygous

TABLE 2 Number of Number of Number of Number of homozygous predictedconfirmed false negative seeds homozygous homozygous homozygousEndosperm identified by seeds that calls based calls based on samplingendosperm did not on leaf endosperm method analysis germinate analysisanalysis Hand 8 out of 36 0 8 (all) 5 (13.9%) Automated 6 out of 24 1 50 Hand 6 out of 36 0 6 (all) 2 (5.6%) Automated 6 out of 24 1 5 0 Hand 5out of 36 0 5 (all) 7 (19.4%) Automated 7 out of 24 2 5 0 Hand 7 out of36 1 6 0 Automated 5 out of 24 2 3 0

Example 2

This example demonstrates the use of the screening methods of thepresent invention in a program for marker-assisted selection of soybeansfor Low Linoleic Acid.

Soybean is the most valuable legume crop, with many nutritional andindustrial uses due to its unique chemical composition. Soybean seedsare an important source of vegetable oil, which is used in food productsthroughout the world. The relatively high level (usually about 8%) oflinolenic acid (18:3) in soybean oil reduces its stability and flavor.Hydrogenation of soybean oil is used to lower the level of linolenicacid (18:3) and improve both stability and flavor of soybean oils.However, hydrogenation results in the production of trans fatty acids,which increases the risk for coronary heart disease when consumed. Thedevelopment of low linolenic acid soybean has been complicated by thequantitative nature of the trait. The low linolenic acid soybeanvarieties that have been developed have been found to yield poorly,limiting their usefulness in most commercial settings. Developing aproduct with commercially significance seed yield is a high priority inmost soybean cultivar development programs.

An example of the application of the screening methods of the presentinvention is selection of soybean plants with both high yield anddecreased linoleic acid content Soybean progeny performance as itrelates to low linoleic acid relies mainly on two major quantitativetrait locus (QTL) at Fad3-1b and Fad3-1c. Analysis of segregating plantsdemonstrated that Fad3-1b and Fad3-1c additively control linoleniccontent in soybean. Therefore, by using a combination of markers forFad3-1b and Fad3-1c, a breeder using the invention can accuratelypredict linolenic acid content in soybean plants. The markers can beused to infer the genotypic state of a seed at any stage in the breedingprocess, for example, at the finished inbred line stage, or the F₁, F₂,F₃, etc.

A seminal F₁ hybrid can be produced by crossing two inbred soybean lines(for example, crossing a plant containing the Fad3-1b and/or Fad3-1calleles associated with decreased linoleic acid content to a plantlacking these alleles) followed by natural self-pollination. Since themarkers can be used to infer the genotypic state of a single seedobtained from an intermating of such inbred lines, early generation(i.e., F₂) marker-assisted breeding can be conducted.

Soybean seed at ambient temperature and humidity typically equilibrateto 8% moisture on a dry weight basis. Soybean seed at this level ofmoisture tends to split when chipped. To reduce splitting, seed shouldbe humidified to moisture level of 12%. When pretreated in this manner,splitting is significantly reduced to <5%.

The selected F₂ seed that have the desired genotype may be bulked orkept separate depending on the breeding objectives. If multiple QTL withvarying effects were being selected from a given population, the breedercould preserve single seed identity to differentiate individuals withvarious combinations of the target resistance QTL. These seeds could beplanted in the field with appropriate field identification. Severalmethods of preserving single seed identity can be used whiletransferring seed from the chipping lab to the field. Methods includetransferring selected individuals to horticultural seed tape that couldalso include radio frequency identification to aid in the identificationof the individual genotyped seed. Other methods would be to use anindexing tray, plant seeds in peat pots and then transplant them, orhand plant from individual seed packets.

Example 3

This example demonstrates the use of the screening methods of thepresent invention in a program for recurrent parent alleles in abackcross breeding program.

The screening methods of the present invention can be used for selectionof transgenes as well as identification of recurrent parent alleles. Theidentification of genotypes with desired recurrent parent allelefrequencies before planting allows the number of rows per population tobe reduced throughout the entire breeding program along with an increasein the number of populations included in the conversion program within agiven field unit. This results in improved land usage, reduced land andlabor costs, etc.

An example of screening endosperm tissue from corn for recurrent parentalleles in a backcross breeding program is shown in FIG. 29.

Example 4

This example demonstrates the use of the screening methods of thepresent invention for use in DNA line fingerprinting and linkage phasedetermination.

Combined with bulking of a single seed's DNA, line fingerprinting couldbe accomplished without the need to sample the line in the field.

By using seed endosperm tissue (seed coat in soybean) derived from adiploid plant, the parental marker haplotypes can be determined using agenotyping system that enables detection of different allele frequenciesin DNA samples. Since endosperm tissue is triploid, with two copiesderived from the female gamete, the linkage phase of the parental linecan be derived by dissecting heterozygous progeny genotypes. The DNAsample from endosperm tissue allows for a determination of the ploidylevel of the genetic marker. A diploid ploidy level in the geneticmarker indicates maternal inheritance and a haploid ploidy level in thegenetic marker indicates paternal inheritance.

1. A sampling system for removing tissue samples from seeds, thesampling system comprising: a hopper for holding a plurality of seeds; aselector for selecting individual seeds from the plurality of seeds inthe hopper; at least one distributor having a plurality of passages forreceiving seeds from the selector; a plurality of seed sampling stationseach configured to receive a seed from the at least one distributor andremove at least one tissue sample from the received seed; a sampletransport system for transporting removed tissue samples away from eachseed sampling station; and a seed transport system for transportingsampled seeds away from each seed sampling station.
 2. The samplingsystem according to claim 1, wherein each seed sampling station isconfigured to remove at least two tissue samples from a seed received bythe seed sampling station.
 3. The sampling system according to claim 1,wherein the sample transport system comprises a tube and nozzle forinducing a pressure differential in the tube to transport the tissuesamples.
 4. The sampling system according to claim 1, wherein the seedtransport system comprises a tube and a nozzle for inducing a pressuredifferential in the tube to transport the sampled seeds.
 5. The samplingsystem according to claim 1, further comprising air nozzles directed toremove tissue samples from the seed sampling stations.
 6. The samplingsystem according to claim 1, wherein the at least one distributorcomprises an outer shell, and a shuttle translatable within the outershell, the outer shell having a plurality of inlet openings, and aplurality of outlet openings, and the shuttle defining the plurality ofpassages therein, the shuttle being operable between a first position inwhich the passages are aligned with the inlet openings to receive seedsthrough the inlet openings, and a second position in which the passagesare aligned with the outlet openings to discharge the seeds from thewells.
 7. The sampling system according to claim 1, wherein the at leastone distributor includes two distributors, each having a plurality ofseparate passages therethrough, and wherein each passage of eachdistributor communicating with a seed sampling station.
 8. The samplingsystem according to claim 1, further comprising at least one sampletray, having a plurality of wells, and a translating mechanism forpositioning each of the plurality of wells into alignment with an outletof the sample transport system.
 9. The sampling system according toclaim 1, further comprising at least one seed tray, having a pluralityof wells, and a translating mechanism for positioning each of theplurality of wells into alignment with an outlet of the seed transportsystem.
 10. The sampling system according to claim 1, wherein theselector comprises a disk having a plurality of openings in its surface,and a vacuum system for creating suction at each of the plurality ofopenings, the suction drawing and releasably holding a seed against thedisk at one of the plurality of openings.
 11. The sampling systemaccording to claim 1, wherein each of the seed sampling stationsincludes a broach having a plurality of cutting edges and configured toremove at least one tissue sample from a seed received in the seedsampling station.
 12. The sampling system according to claim 11, whereinthe broach includes a longitudinal axis, and wherein the plurality ofcutting edges of the broach are aligned generally along the longitudinalaxis of the broach.
 13. The sampling system according to claim 11,wherein each of the seed sampling stations further includes a coringtool configured to remove at least one tissue sample from a seedreceived in the seed sampling station.
 14. The sampling system accordingto claim 11, wherein each of the seed sampling stations further includesa sample frame defining a recess for receiving a seed received from theat least one distributor into the seed sampling station and a samplinggroove into which a portion of the seed received in the recessprotrudes, the sampling system further comprising an extensible supportfor advancing the seed into the recess in the sampling frame, andwherein the broach is positioned and operable to reciprocate in thesampling groove to thereby remove at least one tissue sample from theportion of the seed received in the recess and protruding into thesampling groove.
 15. A sampling system for use in removing tissue fromseeds, the sampling system comprising: a hopper configured to hold aplurality of seeds; a separating wheel configured to singulateindividual seeds from the plurality of seeds in the hopper; a pluralityof seed sampling stations configured to remove tissue from thesingulated seeds, each of the seed sampling stations including a cuttingmechanism configured to progressively remove tissue from a singulatedseed; a plurality of actuators configured to receive the singulatedseeds from the separating wheel and position the singulated seeds in theseed sampling stations, each of the actuators being associated with atleast one of the seed sampling stations and configured to position asingulated seed from the separating wheel adjacent the cutting mechanismof the at least one of the seed sampling stations; a first transportsystem configured to transport removed tissue away from each of the seedsampling stations; and a second transport system configured to transportsingulated seeds away from each of the seed sampling stations aftertissue is removed from the singulated seeds.
 16. The sampling system ofclaim 15, further comprising at least one distributor configured toreceive the singulated seeds from the separating wheel and deliver thesingulated seeds to the actuators.
 17. The sampling system of claim 16,wherein the at least one distributor includes a shaft defining at leastone passage and a sleeve movably disposed at least partially over theshaft, the sleeve defining multiple openings to permit singulated seedsto pass between the separating wheel and the actuators, when at leastone of the openings is aligned with the at least one passage.
 18. Thesampling system of claim 15, wherein each of the seed sampling stationsfurther includes a support configured to receive a singulated seed fromone of the actuators and help hold the singulated seed in the seedsampling station while tissue is removed from the singulated seed. 19.The sampling system of claim 18, wherein the support includes fingersoperable to engage the singulated seed received from said one of theactuators.
 20. The sampling system of claim 15, wherein the cuttingmechanism includes a linear broach having a plurality of cutting edgeslinearly disposed along the broach and configured to remove tissue froma seed when the broach is moved relative to the seed.
 21. The samplingsystem of claim 15, wherein the cutting mechanism is configured to forma kerf in the seed and remove tissue from the seed from within the kerf.22. The sampling system of claim 15, wherein the cutting mechanism isconfigured to remove tissue from the seed from progressively deeperportions of the seed.
 23. The sampling system of claim 15, wherein theseparating wheel includes multiple recesses to capture seeds from thehopper, and wherein each of the recesses are configured to communicatewith a vacuum system to retain the seed in the recess.
 24. The samplingsystem of claim 15, wherein at least one of the seed sampling stationsfurther includes a coring device operable to remove tissue from a seedfrom a kerf formed in the seed by the cutting mechanism.
 25. Thesampling system of claim 15, wherein the first transport system isoperable to deposit the tissue in wells of at least one tray and thesecond transport system is operable to deposit the seeds from whichtissue is removed in wells of at least one tray.
 26. The sampling systemof claim 15, wherein the plurality of seed sampling stations includes atleast six seed sampling stations.